Graduate School of Life and Environmental Sciences, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto, Kyoto 606-8522, Japan.
Medical Research Institute, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima Bunkyo-ku, Tokyo 113-8510, Japan.
J Biochem. 2021 Mar 5;169(2):207-213. doi: 10.1093/jb/mvaa102.
An enzyme, Cut190, from a thermophilic isolate, Saccharomonospora viridis AHK190 could depolymerize polyethylene terephthalate (PET). The catalytic activity and stability of Cut190 and its S226P/R228S mutant, Cut190*, are regulated by Ca2+ binding. We previously determined the crystal structures of the inactive mutant of Cut190*, Cut190S176A, in complex with metal ions, Ca2+ and Zn2+, and substrates, monoethyl succinate and monoethyl adipate. In this study, we determined the crystal structures of another mutant of Cut190, Cut190**, in which the three C-terminal residues of Cut190* are deleted, and the inactive mutant, Cut190**S176A, in complex with metal ions. In addition to the previously observed closed, open and engaged forms, we determined the ejecting form, which would allow the product to irreversibly dissociate, followed by proceeding to the next cycle of reaction. These multiple forms would be stable or sub-stable states of Cut190, regulated by Ca2+ binding, and would be closely correlated with the enzyme function. Upon the deletion of the C-terminal residues, we found that the thermal stability increased while retaining the activity. The increased stability could be applied for the protein engineering of Cut190 for PET depolymerization as it requires the reaction above the glass transition temperature of PET.
一种来自嗜热菌株 Saccharomonospora viridis AHK190 的酶 Cut190 能够降解聚对苯二甲酸乙二醇酯 (PET)。Cut190 及其 S226P/R228S 突变体 Cut190的催化活性和稳定性受到 Ca2+结合的调节。我们之前已经确定了无活性突变体 Cut190S176A 与金属离子 Ca2+和 Zn2+以及底物单乙基琥珀酸酯和单乙基己二酸酯复合物的晶体结构，以及另一种突变体 Cut190的晶体结构，该突变体中 Cut190*的三个 C 末端残基被删除。我们还确定了无活性突变体 Cut190S176A 与金属离子的复合物的晶体结构。除了之前观察到的封闭、开放和结合形式外，我们还确定了推出形式，该形式允许产物不可逆地解离，然后继续进行下一个反应循环。这些多种形式可能是 Cut190 的稳定或亚稳定状态，受到 Ca2+结合的调节，并且与酶功能密切相关。在删除 C 末端残基后，我们发现热稳定性增加，而保持了活性。增加的稳定性可应用于 Cut190 的蛋白质工程，用于 PET 解聚，因为它需要在 PET 的玻璃化转变温度以上进行反应。
